Most marketed formulations for treating anterior ocular conditions are topical, with conventional eyedrops representing the most utilized modality. However, due to the natural clearance mechanisms of the eye, less than 5% of an applied dose remains bioavailable following administration. To overcome the shortcomings associated with conventional eyedrops, a series of enzymatically degradable, mucoadhesive thermogels were developed. Thermogels can be applied as a solution, like a conventional eyedrops, but gel against the heat of eye. To avoid obstructing vision, these thermogels were designed to be instilled within the inferior fornix of the eye. In these studies, the base thermogelling polymer (pNAM) was crosslinked with the natural polymer chitosan. Not only does crosslinking strengthen the typically weak thermogels, but chitosan can be enzymatically degraded by lysozyme, the highest concentration protein found in tear fluid. Therefore, the developed thermogels can be applied to the inferior fornix and degrade over multiple days. A limitation of applying materials to the inferior fornix is they tend to be poorly retained. To anchor the developed thermogels within the inferior fornix, the mucoadhesive properties were tailored based on the chitosan utilized as well as the inclusion of a disulfide monomer capable of covalently bonding with the natural mucosal layer covering the surface of the eye. The disulfide bridging monomer could be further conjugated with therapeutic components which were released as a function of mucosal interaction. Conjugates investigated included cysteamine for treating cystinosis, n-acetyl cysteine for treating dry eye, the adhesion peptide RGDC as a model peptide/protein, and polyethylene glycol for modulating material properties. The release of the drugs Ketotifen Fumarate, for treating allergic conjunctivitis, and atropine, for treating myopia, were also investigated. The safety of the developed thermogels were studied both in vivo and extensively in vitro utilizing both rat and rabbit models. / Dissertation / Doctor of Philosophy (PhD) / Topical eyedrops are the most utilized treatment option for the vast majority of ocular diseases. However, eyedrops are largely ineffective with less than 5% of an applied dose reaching the desired cite of action. Therefore, eyedrops need to be frequently reapplied. To overcome these limitations, an eyedrop was developed which can be applied as a liquid but gels against the heat of the eye. This gel allows for prolonged drug release over multiple days, greatly increasing drug efficacy as well as patient comfort and compliance. To prevent obstructing vision, these gels can be applied under the lower eyelid. To keep these gels retained under the lower eyelid, they were designed to anchor to the natural mucus layer which covers the surface of eye. The developed eyedrops represent a significant advancement in ocular care; bettering the convenience, comfort, and effectiveness for patients of a topical formulation compared to traditional eyedrops.

Nanofluidic devices have been of great interest due to their applications in variety of fields, including energy conversion and storage, water desalination, biological and chemical separations, and lab-on-a-chip devices. Although these applications cross the boundaries of many different disciplines, they all share the demand for understanding transport in nanoscale conduits. In this thesis, different elusive aspects of molecule and particle transport through nanofluidic conduits are investigated, including liquid and ion transport in nanochannels, diffusion- and reaction-governed enzyme transport in nanofluidic channels, and finally translocation of nanobeads through nanopores. Liquid or solvent transport through nanoconfinements is an essential yet barely characterized component of any nanofluidic systems. In the first chapter, water transport through single hydrophilic nanochannels with heights down to 7 nm is experimentally investigated using a new measurement technique. This technique has been developed based on the capillary flow and a novel hybrid nanochannel design and is capable of characterizing flow in both single nanoconduits as well as nanoporous media. The presence of a 0.7 nm thick hydration layer on hydrophilic surfaces and its effect on increasing the hydraulic resistance of the nanochannels is verified. Next, ion transport in a new class of nanofluidic rectifiers is theoretically and experimentally investigated. These so called nanofluidic diodes are nanochannels with asymmetric geometries which preferentially allow ion transport in one direction. A nondimensional number as a function of electrolyte concentration, nanochannel dimensions, and surface charge is derived that summarizes the rectification behavior of this system. In the fourth chapter, diffusion- and reaction-governed enzyme transport in nanofluidic channels is studied and the theoretical background necessary for understanding enzymatic activity in nanofluidic channels is presented. A simple analytical expression that describes different reaction kinetics is derived and confirmed against available experimental data of reaction of Trypsin with Poly-L-lysine. Finally, in the last chapter translocation of nanobeads through synthetic nanopores is experimentally investigated using resistive pulse sensing. The emphasis is placed on elucidating the effect of nanobead size on the translocation current and time. The key goals pursued in this study are multiplex detection of different nanobead sizes in a mixture of nanobeads as well as determining the concentration of each component. This problem other than its fundamental significance paves the way for developing new biosensing mechanisms for detection of biomolecules. This thesis further explores the molecule and particle transport in nanoscale conduits and serves for better characterization and development of nanofluidic devices for various applications.

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24854号 / エネ博第463号 / 新制||エネ||87(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井, 孝, 教授 片平, 正人, 教授 佐川, 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

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Finding new energy sources has been a major quest in the 21st century. The challenge is not simply to find alternatives for traditional energy sources like crude oil and coal to generate power on a large scale but also to produce power to energize micro and nano scale devices. For electricity production in the nano and the micro scale, research has been done on technologies including fuel cells and batteries.This study is focused on two themes, i.e., the use of a microbial fuel cell and an enzymatic fuel cell for potential bioMEMS/NEMS applications. In the first , a fully functional fuel cell with yeast as the microbe using glucose as the fuel was tested to characterize its performance. In the second phase, a lactate dehydrogenase based enzymatic fuel cell using lactate as the fuel was tested to understand the performance characteristics.

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<p>This thesis describes a new type of biosensor, which reports the presence of a target by generating a smell that can be easily detected by the human nose. This approach is radically different from, but complementary to, colorimetric based reporting and it paves the way for the development of multi-sensory biosensors that can be used in a variety of fields, such as diagnostic device, food processing and environmental monitoring</p> <p>Biosensors typically consist of two parts: a bio-recognition element and a signal transducer. The biorecognition element is the component that can specifically interact with its cognate target, while the transducer produces a signal that can be easily identified. The key element of the smell generating biosensor is the enzyme tryptophanase (TPase), which was used as the signal transducer. This enzyme uses either L-tryptophan or S-methyl-L-cysteine as substrates, to produce either indole or methyl mercaptan as final products- both molecules are easily detectable by the human nose. Proof-of-concept for this biosensor was achieved by performing an enzyme-linked immunosorbent assay (ELISA) on magnetic beads with detection of IgG from rabbit serum (the target) in a sample and reporting the presence of the target through the generation of a smell (either indole or methyl mercaptan, depending on the substrate used).</p> <p>The potential use of TPase for biosensing was further expanded by creating a bienzyme system that allows specifically detecting of adenosine-5’-triphosphate (ATP) and reporting its presence by generating a smell. This bienzyme system is based on the fact that TPase activity is greatly affected by the concentration of pyridoxal phosphate (PLP)- which acts as a cofactor that modulates enzyme activity. The enzyme pyridoxal kinase PKase catalyzes the phosphorylation of pyridoxal to PLP in the presence of ATP. The more ATP presents, the more PLP is produced per unit time. If this occurs in the presence of TPase, larger concentrations of ATP in samples will result in higher amounts and faster rates of PLP formation, leading to increased activity of TPase, hence faster generation of either indole or methyl mercaptan is achieved. This bienzyme was used for the detection of DNA molecules with a specific sequence as well as for the detection of microbial cells through smell generation.</p> <p>Most widely used biosensors require immobilization of the biologically active elements on a stable surface. Paper, being a cheap and easy accessible substrate, was used for fabrication of the olfactory-based biosensor. Poly(N-isopropylacrylamide-co-vinylacetic acid) (PNIPAM-VAA) microgels with functional groups present on their surface were modified by biotinylation and loaded with streptavidin/avidin (to be prepared as a platform for further biomolecule immobilization). The microgels were then used as a supporter for the bienzyme system on filter paper to construct a paper-based smell-generating biosensor, which opens the way for the creation of printable smell-reporting printable bio-inks.</p> / Doctor of Philosophy (PhD)

Cell biology is a branch of science with a seemingly infinite abundance of interesting phenomena which are essential to our understanding of life and which may potentially drive the development of technology that improves our lives. Among the open ended questions within the field, an understanding of how gene networks are affected by limited cellular components is both broad and rich with interest. Common to all cellular systems are enzymes which perform many tasks within cells without which organisms could not remain healthy. Here are presented several explorations of enzymatic processing as well as a tool constructed for this purpose. More specifically, these works consider the effect of coupling of gene networks via competition for enzymes found within the cell. It is shown that a limitation on the number of available enzymes permits the formation of bottlenecks which drastically affect molecular dynamics within cells. These effects potentially afford cell behaviors that in part explain the impressive robustness of life to constantly fluctuating environments. / Ph. D.